As used herein, the term "angiogenesis" means the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. The term "endothelium" means a thin layer of flat epithelial cells that lines serous cavities, lymph vessels, and blood vessels.
Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane.
Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops thereby creating the new blood vessel.
Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, tumor metastasis and abnormal growth by endothelial cells and supports the pathological damage seen in these conditions. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic-dependent or angiogenic-associated diseases.
The hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971. (Folkman J., Tumor angiogenesis: Therapeutic implications., N. Engl. Jour. Med. 285:1182 1186, 1971) In its simplest terms it states: "Once tumor `take` has occurred, every increase in tumor cell population must be preceded by an increase in new capillaries converging on the tumor." Tumor "take" is currently understood to indicate a prevascular phase of tumor growth in which a population of tumor cells occupying a few cubic millimeters volume and not exceeding a few million cells, can survive on existing host microvessels. Expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels. For example, pulmonary micrometastases in the early prevascular phase in mice would be undetectable except by high power microscopy on histological sections.
It is clear that angiogenesis plays a major role in the metastasis of a cancer. If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. In the disease state, prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of these diseases.
One example of a disease mediated by angiogenesis is ocular neovascular disease. This disease is characterized by invasion of new blood vessels into the structures of the eye such as the retina or cornea. It is the most common cause of blindness and is involved in approximately twenty eye diseases. In age-related macular degeneration, the associated visual problems are caused by an ingrowth of chorioidal capillaries through defects in Bruch's membrane with proliferation of fibrovascular tissue beneath the retinal pigment epithelium. Angiogenic damage is also associated with diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia. Other diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's sarcoidosis, Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, and corneal graph rejection.
Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales disease, Bechet's disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease, myopia, optic pits, Stargart's disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications. Other diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy.
Another disease in which angiogenesis is believed to be involved is rheumatoid arthritis. The blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to forming new vascular networks, the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. The factors involved in angiogenesis may actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis.
Factors associated with angiogenesis may also have a role in osteoarthritis. The activation of the chondrocytes by angiogenic-related factors contributes to the destruction of the joint. At a later stage, the angiogenic factors would promote new bone formation. Therapeutic intervention that prevents the bone destruction could halt the progress of the disease and provide relief for persons suffering with arthritis.
Chronic inflammation may also involve pathological angiogenesis. Such disease states as ulcerative colitis and Crohn's disease show histological changes with the ingrowth of new blood vessels into the inflamed tissues. Bartonellosis, a bacterial infection found in South America, can result in a chronic stage that is characterized by proliferation of vascular endothelial cells. Another pathological role associated with angiogenesis is found in atherosclerosis. The plaques formed within the lumen of blood vessels have been shown to have angiogenic stimulatory activity.
One of the most frequent angiogenic diseases of childhood is the hemangioma. In most cases, the tumors are benign and regress without intervention. In more severe cases, the tumors progress to large cavernous and infiltrative forms and create clinical complications. Systemic forms of hemangiomas, the hemangiomatoses, have a high mortality rate. Therapy resistant hemangiomas exist that cannot be treated with therapeutics currently in use.
Angiogenesis is also responsible for damage found in hereditary diseases such as Osler-Weber-Rendu disease, or hereditary hemorrhagic telangiectasia. This is an inherited disease characterized by multiple small angiomas, tumors of blood or lymph vessels. The angiomas are found in the skin and mucous membranes, often accompanied by epistaxis (nosebleeds) or gastrointestinal bleeding and sometimes with pulmonary or hepatic arteriovenous fistula.
Numerous efforts have been made by researchers in the pharmaceutical industry to improve the target specificity of drugs. As is familiar to those skilled in the art, the manifestation of a disease many times involves the display of a particular cell type or protein as an antigenic, epitopic, or surface marker. In such instances, an antibody can be raised against the unique cell surface marker and a drug can be linked to the antibody. Upon administration of the drug/antibody complex to a patient, the binding of the antibody to the cell surface marker results in the delivery of a relatively high concentration of the drug to the diseased tissue or organ. Similar methods can be used where a particular cell type in the diseased organ expresses a unique cell surface receptor or a ligand for a particular receptor. In these cases, the drug can be linked to the specific ligand or to the receptor, respectively, thus providing a means to deliver a relatively high concentration of the drug to the diseased organ.
One of the important proteins involved in angiogenesis is ANGIOSTATIN.TM. protein. (see U.S. Pat. No. 5,639,725 to O'Reilly et al., which is incorporated in its entirety by reference herein). ANGIOSTATIN.TM. protein preferably has a molecular weight of between approximately 38,000 Daltons and 45,000 Daltons as determined by reducing polyacrylamide gel electrophoresis, and has an amino acid sequence substantially similar to that of a plasminogen fragment beginning at approximately amino acid number 98 of an intact plasminogen molecule. ANGIOSTATIN.TM. protein has "endothelial inhibiting activity" such that it has the capability to inhibit angiogenesis in general and, for example, to inhibit the growth of bovine capillary endothelial cells in culture in the presence of fibroblast growth factor.
ANGIOSTATIN.TM. protein may be produced from recombinant sources, from genetically altered cells implanted into animals, from tumors, and from cell cultures as well as other sources. ANGIOSTATIN.TM. protein can be isolated from body fluids including, but not limited to, serum and urine. Recombinant techniques include gene amplification from DNA sources using the polymerase chain reaction (PCR), and gene amplification from RNA sources using reverse transcriptase/PCR.
ANGIOSTATIN.TM. protein has been shown to be capable of inhibiting the growth of endothelial cells in vitro and in vivo. ANGIOSTATIN.TM. protein does not inhibit the growth of cell lines derived from other cell types. Specifically, ANGIOSTATIN.TM. protein has no effect on Lewis lung carcinoma cell lines, mink lung epithelium, 3T3 fibroblasts, bovine aortic smooth muscle cells, bovine retinal pigment epithelium, MDCk cells (canine renal epithelium), WI38 cells (human fetal lung fibroblasts), EFN cells (murine fetal fibroblasts) and LM cells (murine connective tissue). Endogenous ANGIOSTATIN.TM. protein in a tumor bearing mouse is effective at inhibiting metastases at a systemic concentration of approximately 10 mg ANGIOSTATIN.TM. protein/kg body weight.
ANGIOSTATIN.TM. protein has a specific three dimensional conformation that is defined by the kringle region of the plasminogen molecule. (Robbins, K. C., "The plasminogen-plasmin enzyme system" Hemostasis and Thrombosis, Basic Principles and Practice, 2nd Edition, ed. by Colman, R. W. et al. J.B. Lippincott Company, pp. 340-357, 1987). There are five such kringle regions, which are conformationally related motifs and have substantial sequence homology in the amino terminal portion of the plasminogen molecule. See FIG. 1 for a schematic diagram of the structure of the plasminogen molecule.
The amino acid sequence of the complete murine plasminogen molecule is shown in SEQ ID NO:81.
A preferred amino acid sequence for human ANGIOSTATIN.TM. protein as shown in FIG. 2.
As used herein, "kringle 1" means a protein derivative of plasminogen having an endothelial cell inhibiting activity, and having an amino acid sequence comprising a sequence homologous to kringle 1, exemplified by, but not limited to that of human kringle 1 corresponding to amino acid positions 11 to 90 (inclusive) of ANGIOSTATIN.TM. protein of SEQ ID NO: 1. As used herein, "kringle 2" is exemplified by, but not limited, to amino acid positions 94 to 172 (inclusive) of ANGIOSTATIN.TM. protein of SEQ ID NO:1. As used herein, "kringle 3" is exemplified by, but not limited to, amino acid positions 185 to 263 (inclusive) of ANGIOSTATIN.TM. protein of SEQ ID NO:1. As used herein, "kringle 4" is exemplified by, but not limited to, amino acid positions 288 to 366 (inclusive) of ANGIOSTATIN.TM. protein of SEQ ID NO:1.
Furthermore, it is understood that a variety of silent amino acid substitutions, additions, or deletions can be made in the above identified kringle fragments, which do not significantly alter the fragments' endothelial cell inhibiting activity, and which are, therefore, not intended to exceed the scope of the claims.
Each kringle region of the plasminogen molecule contains approximately 80 amino acids and contains 3 disulfide bonds. Anti-angiogenic ANGIOSTATIN.TM. protein may contain a varying amount of amino- or carboxy-terminal amino acids from the interkringle regions and may have some or all of the naturally occurring di-sulfide bonds reduced. ANGIOSTATIN.TM. protein may also be provided in an aggregate, non-refolded, recombinant form. Additionally, individual and groups of kringle peptides may be useful for inhibition of angiogenesis (see PCT/US96/05856, which is incorporated herein by reference).
The cDNA sequence for human ANGIOSTATIN.TM. protein is provided as SEQ ID NO:29.
It is contemplated that any isolated protein or peptide having a three dimensional kringle-like conformation or cysteine motif that has anti-angiogenic activity in vivo, is also an ANGIOSTATIN.TM. protein compound. The amino acid sequence of the ANGIOSTATIN.TM. protein of the present invention may vary depending upon, for example, from which species the plasminogen molecule is derived. Thus, although the ANGIOSTATIN.TM. protein of the present invention that is derived from human plasminogen has a slightly different sequence than ANGIOSTATIN.TM. protein derived from mouse, it has anti-angiogenic activity as shown in a mouse tumor model.
Another important angiogenesis-related protein is ENDOSTATIN.TM. protein. (see U.S. Pat. No. 5,854,25 and WO 97/15666 O'Reilly et al., both of which are incorporated in their entirety by reference herein) ENDOSTATIN.TM. protein is a potent and specific inhibitor of endothelial proliferation and angiogenesis. Systemic therapy with ENDOSTATIN.TM. protein causes a nearly complete suppression of tumor induced angiogenesis.
ENDOSTATIN.TM. protein has a molecular weight of approximately 18,000 to approximately 20,000 Daltons as determined by non-reduced and reduced gel electrophoresis, respectively, and is capable of inhibiting endothelial cell proliferation in cultured endothelial cells. ENDOSTATIN.TM. protein has an amino acid sequence substantially similar to a fragment of a collagen molecule and whereas it binds to a heparin affinity column, it does not bind to a lysine affinity column.
ENDOSTATIN.TM. protein can be isolated from murine hemangioendothelioma EOMA. ENDOSTATIN.TM. protein may also be produced from recombinant sources, from genetically altered cells implanted into animals, from tumors, and from cell cultures as well as other sources. ENDOSTATIN.TM. protein can be isolated from body fluids including, but not limited to, serum and urine. Recombinant techniques include gene amplification from DNA sources using the polymerase chain reaction (PCR), and gene amplification from RNA sources using reverse transcriptase/PCR.
Alternatively, endothelial proliferation inhibiting proteins, or ENDOSTATIN.TM. proteins, of the present invention may be isolated from larger known proteins, such as human alpha 1 type XVIII collagen and mouse alpha 1 type XVIII collagen, proteins that share a common or similar N-terminal amino acid sequence. Examples of other potential ENDOSTATIN.TM. protein source materials having similar N-terminal amino acid sequences include Bos taurus pregastric esterase, human alpha 1 type XV collagen, NAD-dependent formate dehydrogenase (EC 1.2.1.2) derived from Pseudomonas sp., s11459 hexon protein of bovine adenovirus type 3, CELF21D12 2 F21d12.3 Caenorhabditis elegans gene product, VAL1 TGMV AL1 protein derived from tomato golden mosaic virus, s01730 hexon protein derived from human adenovirus 12, and Saccharomyces cerevisiae.
Human ENDOSTATIN.TM. can be further characterized by its preferred amino acid sequence as set forth in FIG. 3 and in SEQ ID NO: 2. The preferred sequence of the first 20 amino-terminal amino acids corresponds to a C-terminal fragment of collagen type XVIII or collagen type XV. Specifically, in one embodiment the amino terminal amino acid sequence of ENDOSTATIN.TM. protein corresponds to an internal 20 amino acid peptide region found in mouse collagen alpha 1 type XVIII starting at amino acid 1105 and ending at amino acid 1124. The amino terminal amino acid sequence of the inhibitor also corresponds to an internal 20 amino acid peptide region found in human collagen alpha 1 type XVIII starting at amino acid 1132 and ending at amino acid 1151. The cDNA sequence for ENDOSTATIN.TM. protein is provided as SEQ ID NO: 30.
Both ANGIOSTATIN.TM. protein and ENDOSTATIN.TM. protein specifically and reversibly inhibit endothelial cell proliferation and may be used, for example, as a birth control drug, for treating angiogenesis-related diseases, particularly angiogenesis-dependent cancers and tumors, and for curing angiogenesis-dependent cancers and tumors. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of such diseases mediated by angiogenesis. Potential therapies useful for controlling angiogenic processes may involve recognition of antigenic markers and receptors associated with angiogenesis and subsequent modification of such markers and receptors. For example, once a receptor for an angiogenesis-related protein is identified, it can be blocked, thereby inhibiting the effect of the angiogenesis-related protein and ultimately reducing angiogenesis.
One technique that is useful for identifying antigenic markers and receptors is phage-display technology. (see for example Phage Display of Peptides and Proteins: A Laboratory Manual. Edited by Brian K. Kay et al. Academic Press San Diego, 1996) Phage-display technology is a powerful tool for the identification of individual epitopes that interact with ligands such as proteins and antibodies. Phage peptide libraries typically comprise numerous different phage clones, each expressing a different peptide, encoded in a single-stranded DNA genome as an insert in one of the coat proteins. In an ideal phage library the number of individual clones would be 20.sup.n, where "n" equals the number of residues that make up the random peptides encoded by the phage. For example, if a phage library was screened for a seven residue peptide, the library in theory would contain 20.sup.7 (or 1.28.times.10.sup.9) possible 7-residue sequences. Therefore, a 7-mer peptide library should contain approximately 10.sup.9 individual phage.
Phage clones displaying peptides that are able to mimic epitopes recognized by a particular protein (or antibody), are selected from the library based upon their binding affinity to that protein (or antibody), and the sequences of the inserted peptides are deduced from the DNA sequences of the phage clones. This approach is particularly desirable because no prior knowledge of the primary sequence of the target protein is necessary, epitopes represented within the target, either by a linear sequence of amino acids (linear epitope) or by the spatial juxtaposition of amino acids distant from each other within the primary sequence (conformational epitope) are both identifiable, and peptidic mimotopes of epitopes derived from nonproteinaceous molecules such as lipids and carbohydrate moieties can also be generated.
With regard to angiogenesis-related disease, it is evident that angiogenesis-related proteins such as ANGIOSATIN.TM. protein and ENDOSTATIN.TM. protein play an important role in the development of disorders such as cancer. What is needed therefore, is the development of methods and compositions for the identification of receptors and molecules that bind such proteins. The identification of such receptors and molecules would facilitate the understanding of angiogenesis-related protein influence and interaction, and consequently enable the development of drugs to modify the activity of these proteins as necessary.